The vertebrate central nervous system (CNS) is composed of a wide variety of neurons that are generated following tightly-regulated developmental programs. Characterization of the function and specificity of molecules and regulatory elements working on distinct neuronal populations is thus essential in order to enhance our understanding of how such complexity is achieved in the developing brain and how it is maintained in the adult brain.
Cholesterol is present at high levels in the CNS of vertebrates and is metabolized in the brain, predominantly to 24S-hydroxycholesterol (24S-HC). Neurodegenerative conditions that occur are as a result of neurons in the brain being lost. In conditions such as Parkinson's disease, which is a common neurodegenerative disease, the condition is linked to the loss of substantial nigra midbrain dopaminergic neurons. The loss of nigrostriatal neurons results in symptoms such as tremors that are a classic symptom of the illness.
Research has worked on the use of cell replacement therapy (CRT) and regenerative medicine to try and combat the disease, which is becoming more prevalent as populations age. However, an alternative means of developmental and adult regulation is via nuclear receptors. It has been found that the liver X receptor (Lxr) ligand is a specific inducer of midbrain dopaminergic neurons both in embryonic stem cells, neural tissues and even in whole animals. In particular, examples of nuclear receptors expressed in embryonic and adult brain having both a developmental role and a function in the adult brain are the liver X receptors (Lxrα and β). The liver X receptors (Lxrα and β), are activated by oxysterols. Analysis of double Lxrα and Lxrβ knockout mice revealed that Lxrs are required for neurogenesis during ventral midbrain (VM) development. Moreover, adult male Lxrβ knockout mice (Lxrβ−/−) show a progressive accumulation of lipids in the brain and loss of spinal cord motor neurons, suggesting a neuroprotective role of Lxrs and their ligands on adult motor neurons. Similarly, the number of Islet1+ oculomotor neurons is lower in the developing midbrain of Lxrα−/−β−/− mice, indicating a role of Lxrs, not only in the maintenance of adult motor neurons, but also in their development. Enzymes involved in the synthesis of cholesterol and oxysterols, such as 2,3-oxidosqualene-lanosterol cyclase, are localized in Islet1+ oculomotor neurons in the mouse ventral midbrain and it has been found that oxysterols and endogenous brain Lxr ligands are sufficient to regulate neurogenesis in the developing ventral midbrain. While endogenous brain Lxr ligands have been identified and found to regulate the development of midbrain dopamine neurons and red nucleus neurons (Theofilopoulos et al. (2012) Nat. Chem. a Biol. 9, 126-133), to date, no endogenous ligand that is capable of regulating the survival of motor neurons in vivo has so far been identified.
It has also been reported that cholesterol metabolites that had the capacity to activate Lxrs can be identified in human cerebrospinal fluid (CSF) (Ogundare M, et al. J Biol Chem 2010; 285(7):4666-79.).
In order to identify novel Lxr ligands that regulate motor neuron function the applicants delved deeper into the human CSF sterolome and examined plasma of patients with different human diseases associated with motor neuron degeneration, hereditary spastic paresis (HSP) type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX) as well as infants with oxysterol 7α-hydoxylase deficiency (O7AHD). These diseases result from mutations in the cytochrome P450 CYP7B1 (SPG5 and O7AHD) and CYP27A1 genes (CTX). The enzymes coded by these genes are responsible for 7α-hydroxylation of oxysterols and (25R),26-hydroxylation of sterols, respectively, reactions that generate further oxysterols and ultimately cholestenoic acids (FIG. 1 hereinafter). The applicants found that, surprisingly, specific cholestenoic acids with a 3β-hydroxy-5-ene, but not a 3-oxo-4-ene, structure activate Lxrα and β in neuronal cells, increase expression of Islet-1, a transcription factor required for the development of motor neurons, and promote the survival of Islet1+ oculomotor neurons. Moreover these effects were abolished by knock-down or knock-out of the Lxrs in zebrafish or in rodent models.
In addition, the applicants showed that patients with CTX, SPG5 and O7AHD are unable to synthesize normal amounts of the Lxr ligand 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA), a cholestenoic acid that the applicants found promotes neuronal survival. This is of interest in relation to the fact that patients with SPG5 present with motor neuron degeneration and spastic paraplegia. Patients with CTX may sometimes also present with spasticity, possibly due to upper motor neuron degeneration. These results have important implications for the treatment neurological diseases leading to motor neuron degeneration. They indicate that cholestenoic acid acting as Lxr ligands, as well as inhibitors of specific biosynthetic enzymes in the cholestenoic acid biosynthetic and metabolic pathways, are useful pharmaceuticals for the treatment of motor neuron disorders.
Furthermore, the applicants showed that whilst patients with CTX had abnormally low levels of all of cholest-(25R)-5-en-3β,26-diol 26-HC (26-HC), 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) and 3β,7β-dihydroxycholest-5-en-26-oic (3β,7β-diHCA), patients with SPG5 and O7AHD had elevated levels of some or all of these (FIG. 1) and that 3β-HCA and 3β,7β-diHCA, in particular, decreased neuronal survival.
All of these results have important implications for the diagnosis of neurological diseases leading to motor neuron degeneration. They indicate that the levels of certain cholestenoic acids (3β-HCA, 3β,7β-diHCA and 3β,7β-diHCA) and a precursor (26-HC) alone or in combination are diagnostic and/or prognostic for motor neuron degenerative disease and/or the level of neurodegeneration.